DESCRIPTION (Applicant's Abstract): The long term goal of this proposal is to define the interactions between tight junction (TJ) proteins and the factors regulating their activity in the lung. Four central questions in TJ biology are addressed. 1. What is the role of occludin, claudin-1 and ZO-1 in regulating the barrier function of TJs? This is assessed using unique inducible, doubly transfected epithelial cell clones to vary the relative expression of these proteins; the effect on TJ permeability is then examined. 2. How do specific domains of the claudins determine TJ barrier function? To examine this, specific amino acid sequences are deleted from cytoplasmic and extra-cellular domains of claudin-1 or -4. The TJ permeability properties of epithelial cell clones expressing these modified proteins are evaluated. 3. How is the profound effect of cholesterol (CH) efflux on TJ function related to detergent insoluble CH/glycolipid rafts and is CH closely associated with TJ proteins? A novel radiolabeled, photoactivatable CH analog is used to determine whether CH is bound to occludin and/or the claudins. Differences in the proportions of integral TJ proteins and the lipid composition of the TJ rafts in high and low resistance strains of epithelial cells will provide new clues as to their role in TJ permeability. Increased lipid 2M messenger generation by activation of phospholipases during CH efflux suggest that they are important regulators of TJ barrier function. These phospholipases will be identified and characterized. 4. To what extent are the permeability properties of pulmonary TJs dictated by their protein composition? Immunogold labeling of occludin and six lung-associated claudins is used to map their location to specific epitheliall endothelial TJs of the lung. An LPS-induced model of acute inflammation is used to explore how TJ proteins and the associated lipid rafts, isolated from airway epithelium, are modified during migration of inflammatory cells. The proposed studies will provide new insights into TJ biology and allow development of new strategies to regulate passage of therapeutic agents across lung epithelia and/or to prevent the penetration of allergens through the TJ barrier.